Detection of point mutations associated with resistance of Helicobacter pylori to clarithromycin by hybridization in liquid phase

Abstract

When the standard procedure for determining antibiotic susceptibility of bacteria is used, the results are delayed, especially for bacteria that grow slowly, such as Helicobacter pylori. Treatment for this bacterium may involve clarithromycin, a compound for which resistance has been associated with point mutations on the 23S rRNA gene. This resistance is currently found in organisms isolated from 0 to 15% of patients and jeopardizes the success of the treatment. We have designed a test involving amplification and colorimetric hybridization in the liquid phase to detect the mutation at the molecular level. First, four reference strains, including the wild type and three strains with the mutations A2143C, A2143G, and A2144G, were used to optimize the method. Amplification was carried out with primers previously published. The amplified products were added to probe-coated microtiter wells. A DNA enzyme immunoassay was used to detect the hybrids. The optimal conditions of the hybridization were defined for each probe. Nineteen H. pylori strains resistant to clarithromycin and 22 susceptible according to phenotypic data were submitted to restriction with BsaI and BbsI, and part of the 23S rRNA gene was sequenced in order to determine the mutation involved for the resistant strains. The new assay showed a complete correlation with the reference methods, except for one strain. Cross-hybridizations as well as application of the reaction to other bacteria did not lead to optical densities higher than the cutoff values chosen with the receiving operating characteristic curve. This method can be easily standardized and gives a result within a day. Its application directly to the biopsy specimens or infected gastric juice is planned in the future.

FIG. 1

Effect of temperature of hybridization on the intensity of the signal detected. The p43G probe was hybridized with its complementary sequence (com43G) at various temperatures (48, 49, and 50°C) and for three probe concentrations (0.5, 1, and 2 ng/μl). The com43G concentration was 1 ng/μl.

FIG. 2

Determination of an optimal probe concentration by using synthetic oligonucleotides. Each probe was hybridized with its complementary sequence (1 ng/μl) at various concentrations of between 0 and 2 ng/μl.

FIG. 3

ROC curves obtained for the probes corresponding to the wild-type genotype (pwt) (A), the A2143G genotype (p43G) (B), and the A2144G genotype (p44G) (C), with percent sensitivity values (true-positive rates) plotted on the y axis and the complementary percent specificity values (true-negative rates) plotted on the x axis.